Process for the production of 6-keto-17alpha-methyltestosterone



PROCESS FoR THEPRODUCTION F euro-17- MQETHYLTESTDSTERONE Herbert Hickory Corners, and Durey H. Peterson, Kalamazoo, Mich., assignors to The Upjohn Company, Kalamazoo, Mich a corporation of Michigan ing structural formula:

OH CH1 L-: -CH;

and may bev prepared by the selective oxidation of the 6fi-hydroxy group of 6fl-hydroxy-l7a-methyltestosterone to a 6-keto group with an oxidizing agent such as chnomic acid, e. g., at least about two equivalents of chromic acid.

It is an object of this invention to provide 6-keto'-,17umethyltestosterone and a convenient method for its. production from readily available 6Brhydr0xy-17wmethyltestos'terone. Other objects will be apparent to those skilled in the art to which this invention pertains.

The novel compound of this invention, 6-keto-l7 methyltestosterone, is not only a pharmacologically and physiologically active compound possessing essentially protein anabolic, adrenocorticotropic hormonal and anesthetic proper-ties, but is also a valuable intermediate for the synthesis of other steroids. For'ex-ample, dehydration of 6-keto-17a-methyltestosterone with phosphorus oxychloride and ozoniza-tion of the thus-obtained 6-keto- 17-methylene-4-androstene-3,6-dione yields 4-androstene- 3,6,l7-trione [Butenandt, Ber. 69, 1163 (1936)] which possesses estrogenic activity.

The starting material of the present invention is 618- hydroxy-17a-methyltestosterone which may be prepared by biooxidation of 17 a-methyltestosterone with the aid of ,a mold of the Rhizopus nigricans strain as shown in "Preparation 1.

In carrying out the process of the present invention, 63- hydroxy-17m-methyltestosterone, dissolved in an organic solvent such as acetic acid, benzene, toluene, petroleum ether, hexanes (Skellysolve B), dioxane or similar solvents or mixtures thereof, with acetic acid and benzene being preferred, is oxidized with a solution containing chromic acid. The chromic acid may be added as chromic acid anhydride together with a small, but sufficient amount of water to produce the dichromate ion (Cr2Or), or

may be produced in situ by reaction between an alkali- 7 Patented Nov. 20, 1956 metal dichromate such as sodium or potassium dichro-.

mate and an acid, for example, acetic acid, formic acid, or sulfuric acid. The reaction may be carried out in a heterogeneous or a homogeneous system. If the reaction is carried out in ajheterogeneous system, 6/3-hydroxy- 17a-methyltestosterone, dissolved in anorganic solvent which is inert to oxidation under the reaction conditions, such as benzene, chlorobenzene, brom-obenzene, hexane, chloroform, halogenated hydrocarbons, ethyl acetate, or

a similar solvent, is admixed with a solution of aqueous sodium dichromate or potassium dichromate acidified with sulfuric acid, or sulfuric acid combined with acetic acid. Vigorous agitation is then employed to bring the organic layer into intimate contact with the aqueous solution. The reaction time is mainly dependent on eflicient stirring. In the preferred embodiment of the invention, the

' amounts of water with chromic .anhydride. At the termito about 24 hours or even longer.

nation of the reaction, excess of chromic acid may be destroyed by adding methyl or ethyl alcohol to the solution and concentrating the solution on a steam bath in vacuo. The temperature of the reaction is maintained between about zero and about fifty degrees centigrade, with temperatures between about fifteen and thirty degrees centigrade preferred. The reaction time depends on the temperature and may vary between about one hour The thus-obtained 6- keto-17a-methyltestosterone is isolated from the reaction mixture by extraction with organic solvents, for example, ether, ethyl acetate, chloroform, methylene dichloride or water-immiscible alcohols and purified by conventional means, such as recrystallization and chromatography.

The. following examples are illustrative of the process and product of the present invention, but are not to be construed as limiting.

PREPARATION l.6fl-HYDROXY-17ot-METHYLTESTOSTERONE A was prepared of twenty grams of Edamine enzymatic digest of lactalbum-in, three grams of corn steep liquor and fifty grams of technical dextrose diluted to one liter with'water and adjusted to a pH of 4.3 to 4.5. Twelve liters of this sterilized medium was inoculated with Rhizopus nigricans minus strain, American Type Culture Collection Number 6227b, and incubated for 24 hours at a temperature of 28 degrees centigrade using a rate of aeration and stirring such that the oxygen uptake was 6.3 to 7 millimoles per hour per liter of NflaSOs according to the method of Cooper, Ferns'trom and Miller [Ind. Eng. Chem. 36, 504 (1944)]. To this medium containing a 24-hour growth of Rhizopus nigricans minus strain was added six grams Una-methyltestosterone in 120 milliliters of absolute ethanol to pro- After an additional 24-hour period of incubation under the same conditions of temperature and aeration, the beer and mycelium were extracted. The mycelium was filtered, washed twice, each time with a volume of acetone approximately equal to the volume of the mycelium and extacted twice, each time with a volume of methylene chloride approximately equal to the volume of the mycelium. The acetone and methylene chloride extracts including solvent were added to the beer filtrate. The

\ I mixed extracts and beer filtrate were extracted successively carbonate and then with two one-tenth by volume portions of water. After drying the methylene chloride with about three to five grams of anhydrous sodium sulfate per liter of solvent and filtering, the solvent was. removed by distillation. The extractives obtained. upon-evaporation of the methylene chloride solvent were taken. up in benzene to leave a residue of benzene insoluble'crystals. These were washed with additional benzene to give 550 milligrams of white crystals. Recrystallization from a mixture of equal parts of ethyl acetate and acetone gave 500 milligrams of crystals melting at 220 to 235 degrees centigrade. These were redissolved in three milliliters of methanol and ether was added to reprecipitate the crystals. The. resulting crystals of 6B-hydroxy-17wmethyltestosterone weighed 212 milligrams and had a. melting point of 247 to 252 degrees centigrade. Infrared and ultraviolet spectra verified the structure.

Example 1.?6-ket0-1'7a-methyltestosterone A solution of fifty milligrams of 6B-hydroxy-l7amethyltestosterone, dissolved in one milliliter. of acetic acid, was admixed at zero degrees centigrade with a solution of 15.7 millignams of'chromium trioxide in a mixture of four milliliters of glacial acetic acid and 0.02 milliliter of water. The reaction mixture was kept at room temperature, about twenty to 25 degrees centigrade, for a period of six-teen hours. At the completion of this period, ten drops of methanol and 45 milliliters of water were added and, after standing for ten minutes at room temperature, the mixture was extracted with three fifteenmillili-ter portions of methylene dichloride. The combined extracts were washed with the following ten-milliliter portions: four times with saturated sodium bicar-v bonate solution and two times with water. The ethermethylene dichloride extracts were then dried over anhydrous sodium sulfate and evaporated to give 62 milligrams of solids. These solids were dissolved in six milliliters of benzene and chromatographed over a column containing three grams of alumina previously activated by treatment with acids and drying at 1'20 degrees centigrade; The column was developed with six-milliliter volumes of solvents as follows:

Table I Fraction Solvent Eluate Solids,

Milligrams benzene Fractions 21 and 22, having a combined weight of 12.6 milligrams, were found to be 6-keto-l7a-methyltestosterone. Infrared analysis confirmed the structure of the product as 6-keto-17u-methyltes'tosterone.

Example 2.6-ket0-17a-methyltestoster0ne A solution of fl-hydroxy-l7u-methyltestosterone in benzene was agitated for twelve hours with an aqueous solution of sodium dichromate and dilute sulfuric acid at room temperatures. The benzene layer was separated from the aqueous. solution, washed, dried over anhydrous sodium sulfate and evaporated to yield 6-keto-l7a-metl1yltestosterone which was purified by chromatography as shown in Example 1.

Example 3.-6 -ket0-1 7 a-methyltestosterone A solution of 100 milligrams of 6/3-hydroxy-17oc-methyltestosterone in a mixture of two milliliters of benzene and one milliliter of glacial acetic acid was admixed. at zero. degrees centigrade with a solution of sixty milligrams of sodium dichromate in a mixture of 0.7 milliliter of glacial acetic acid and 0.2 milliliter of benzene. The solution was vigorously stirred for a period of six hours, while kept in an ice-bath. At the completion of this period, 35 milliliters of water was added and the mixture was extracted with three twenty-milliliter portions of a solvent mixture consisting of ether-methylene dichloride in a one to one ratio. The combined extracts were washed with the following ten-milliliter portions: three times with three percent hydrochloric acid solution, twice with water, twice with four percent potassium hydroxide, and twice with water. The ether-methylene dichloride extracts were then dried over anhydrous sodium sulfate, evaporated and the solids were chromatographed as shown in Example 1 and recrystallized from acetone and methan-ol to give 6-keto-l7a-methyltes'tosterone.

It is to be understood that the invention is not to be limited to the exact details of operation shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art, and the invention is therefore to be limited only by the scope of the appended claim We claim:

A process for the production of 6-keto-17a-methyltestosterone which comprises: contacting 6fl-hydroxy-17emethyltestosterone with chromic .acid at a temperature between fifteen and thirty degrees centigrade and separating the thus-produced 6-keto-17a-methyltestosterone.

References Cited in the file of this patent Chem; Abst. 46: cols. 11375-76 (1942}, Abstracting an-article ofDevis in'Acta Chim. Belg. 6, 525-632 (1951'). 

